1. Technical Field
The present invention relates to apparatus and methods for analysis of blood samples in general, and for the determination of hemoglobin related parameters of a blood sample in particular.
2. Background Information
Physicians, veterinarians and scientists have examined human and animals' biologic fluids, especially blood, in order to determine constituent particulate quantities as well as to identify the presence of unusual particulates not seen in healthy subjects. The particulates generally measured, quantified and identified include red blood cells (RBCs), white blood cells (WBCs), and platelets. RBC analyses can include determinations of RBC number, size, volume, shape, hemoglobin content and concentration, and the hematocrit (also referred to as the packed cell volume). RBC analyses can also involve determining the presence and/or concentration of certain components within the red cells such as DNA, RNA, including the detection of the presence and/or enumeration of hematoparasites (e.g., malarial parasites) either in the RBCs or trypanosomes which are extracellular or leishmaniasis organisms which are in the WBCs as well as many other hematoparasites. WBC analyses can include a determination of the population frequency of WBC sub types generally referred to as a differential WBC count, as well as the notification of any unusual cell types not found in healthy subjects. Platelet (or in certain animals including birds, reptiles and fish, thrombocytes which are similar in function to platelets in mammals but are about ten times larger and nucleated) analyses can include platelet number, size, shape texture, and volume determinations, including determining the presence of clumps of platelets or thrombocytes within the sample.
The amount of information that can be determined by examining the blood of a human or animal is vast. It is particularly useful to determine the indices of RBCs; e.g., individual cell size, individual cell hemoglobin content and concentration, and population statistics of RBCs within a sample. The mean and dispersion statistics (e.g., coefficients of variation) for each of the aforementioned parameters can also provide important information, which has enabled physicians to better categorize disorders of RBCs.
Hemoglobinometry is very often performed on a sample by interrogating the sample with light. The amount of absorbance associated with hemoglobin varies considerably as a function of the wavelength of the interrogating light, as can be seen in FIG. 1, which illustrates absorbance versus wavelength for three separate RBCs. Hemoglobinometry is generally performed on precisely diluted blood, where the red blood cells are lysed and where the hemoglobin released from the lysed red blood cells is chemically modified (e.g., methods that convert it to cyanmethemoglobin or azo-methemoglobin) so that its molar extinction coefficient at wavelengths where hemoglobin has a significant and useful absorption are not affected by the ratio of oxyhemoglobin and deoxyhemoglobin. If the molar extinction coefficient is accurately known, the concentration of the hemoglobin in a sample can be accurately determined. In hemoglobin that is not chemically altered, however, the relative molar extinction coefficients of the spectra at useable wavelengths can differ by as much as 10 to 15 percent. For example, the graph shown in FIG. 2 shows the molar extinction coefficient for deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) as a function of wavelength. It is clear from FIG. 2 that the absorbance spectrums for the two forms of hemoglobin (Hb, HbO2) differ significantly. Consequently, an error in the molar extinction coefficient value can introduce significant error into the calculations of related blood indices.
Prior art has addressed this problem by chemically modifying the blood sample, or by evaluating an unmodified blood sample using simultaneous equations for absorption over two different wavelengths, which adds expense and complexity to the process. The isosbestic points (i.e., points where the two species of hemoglobin have identical molar absorptivity) are not as sensitive or useful in clinical measurement due to their lack of flatness and relative low absorptivity.